DSD-Guided Ultrasound-Assisted Protein Recovery From Chlamydomonas reinhardtii: Modeling, Optimization, and Enzyme Inhibitory Activity Assessment.

Chlamydomonas reinhardtii is an emerging protein-rich microalga with significant potential as a sustainable source of bioactive peptides. In this study, an ultrasound-assisted alkaline extraction was developed and optimized using a deterministic screening design (DSD) combined with design space analysis to establish a mechanistic and statistical co-optimization strategy for efficient protein recovery. A total of 15 experimental runs were conducted to systematically evaluate six key variables, including solution pH, solid-to-liquid ratio, extraction temperature, extraction time, ultrasonic power, and ultrasonication duration, in order to identify significant factors affecting extraction yield and construct a predictive regression model. Under optimized conditions, the protein extraction yield reached 78.29% ± 0.98%, and the model exhibited excellent fitting and predictive performance (R2 = 0.993, R2_pred = 0.993, p < 0.001). The recommended operating ranges determined by design space analysis were NaOH solution pH 8.3-9.1, solid-to-liquid ratio 1:152-1:162, and ultrasonic power 310.65-390.65 W, with a verified target achievement probability exceeding 95%. The extracted proteins demonstrated strong antioxidant activity and exhibited concentration-dependent inhibitory effects against α-amylase and α-glucosidase. Enzymatic hydrolysis with seven proteases improved inhibitory potency; proteinase K and bromelain hydrolysates performed best. Their α-amylase IC50 values were 4.97 ± 0.24 and 8.68 ± 0.12 mg/mL, and α-glucosidase IC50 values were 2.15 ± 0.11 and 4.33 ± 0.23 mg/mL, respectively. These findings indicate that DSD-guided optimization effectively improves protein extraction efficiency from C. reinhardtii, and the resulting hydrolysates possess promising hypoglycemic potential for functional food applications. Future studies should focus on identifying specific bioactive peptide sequences and elucidating their enzyme inhibition mechanisms. PRACTICAL APPLICATIONS: An efficient and flexible microalgae protein extraction method was developed using design space optimization, defining a range of conditions rather than a single point for greater practicality. The extracted proteins produce bioactive peptides with potential blood sugar-regulating effects, offering a sustainable, high-value ingredient for functional foods or nutraceuticals.